Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>

ABSTRACT Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and t...

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Autores principales:	Pattarana Sae-Chew, Thidarat Rujirawat, Yothin Kumsang, Penpan Payattikul, Tassanee Lohnoo, Wanta Yingyong, Chalisa Jaturapaktrarak, Tiwa Rotchanapreeda, Onrapak Reamtong, Tanawut Srisuk, Weerayuth Kittichotirat, Theerapong Krajaejun
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Pythium insidiosum cell-free protein synthesis evolution immunoreactive protein oomycete pythiosis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4ae435dab2cb4a8ea971a28d91e4d976
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spelling	oai:doaj.org-article:4ae435dab2cb4a8ea971a28d91e4d9762021-12-02T18:44:35ZAutomated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>10.1128/mSystems.00196-202379-5077https://doaj.org/article/4ae435dab2cb4a8ea971a28d91e4d9762020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00196-20https://doaj.org/toc/2379-5077ABSTRACT Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica. The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen. IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.Pattarana Sae-ChewThidarat RujirawatYothin KumsangPenpan PayattikulTassanee LohnooWanta YingyongChalisa JaturapaktrarakTiwa RotchanapreedaOnrapak ReamtongTanawut SrisukWeerayuth KittichotiratTheerapong KrajaejunAmerican Society for MicrobiologyarticlePythium insidiosumcell-free protein synthesisevolutionimmunoreactive proteinoomycetepythiosisMicrobiologyQR1-502ENmSystems, Vol 5, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Pythium insidiosum cell-free protein synthesis evolution immunoreactive protein oomycete pythiosis Microbiology QR1-502
spellingShingle	Pythium insidiosum cell-free protein synthesis evolution immunoreactive protein oomycete pythiosis Microbiology QR1-502 Pattarana Sae-Chew Thidarat Rujirawat Yothin Kumsang Penpan Payattikul Tassanee Lohnoo Wanta Yingyong Chalisa Jaturapaktrarak Tiwa Rotchanapreeda Onrapak Reamtong Tanawut Srisuk Weerayuth Kittichotirat Theerapong Krajaejun Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>
description	ABSTRACT Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica. The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen. IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.
format	article
author	Pattarana Sae-Chew Thidarat Rujirawat Yothin Kumsang Penpan Payattikul Tassanee Lohnoo Wanta Yingyong Chalisa Jaturapaktrarak Tiwa Rotchanapreeda Onrapak Reamtong Tanawut Srisuk Weerayuth Kittichotirat Theerapong Krajaejun
author_facet	Pattarana Sae-Chew Thidarat Rujirawat Yothin Kumsang Penpan Payattikul Tassanee Lohnoo Wanta Yingyong Chalisa Jaturapaktrarak Tiwa Rotchanapreeda Onrapak Reamtong Tanawut Srisuk Weerayuth Kittichotirat Theerapong Krajaejun
author_sort	Pattarana Sae-Chew
title	Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>
title_short	Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>
title_full	Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>
title_fullStr	Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>
title_full_unstemmed	Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>
title_sort	automated cell-free multiprotein synthesis facilitates the identification of a secretory, oligopeptide elicitor-like, immunoreactive protein of the oomycete <named-content content-type="genus-species">pythium insidiosum</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/4ae435dab2cb4a8ea971a28d91e4d976
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Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete <named-content content-type="genus-species">Pythium insidiosum</named-content>

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